1. Field of the Invention
The present invention relates to a particle agglutination-evaluating container for use in evaluation of a particle agglutination pattern in an immunological agglutination reaction. In particular, the present invention relates to a particle agglutination-evaluating container for evaluation of various blood types or detection of an antigen or antibody, based on the agglutination pattern of blood cell particles.
2. Description of the Related Art
Conventionally, immunological agglutination reactions such as antigen-antibody reactions have been examined using a test tube as the reaction container. In such an examination, antigen-bound latex particles or antigen-presenting red blood cell particles and an antigen-reactive antibody are mixed in a test tube to form a particle-antibody agglutinate; the agglutinate is allowed to precipitate spontaneously or by centrifugation; and then the precipitate is loosened by shaking the test tube to evaluate the agglutination reaction. If the precipitate is not loosened, the reaction is evaluated as positive, and if loosened, it is evaluated as negative. However, in the examination method, it is difficult to identify an intermediate reaction (weakly positive reaction) between the positive and negative reactions with the naked eye. In addition, when a large number of samples are examined, the examination method requires an increased number of test tubes and thus the operation is troublesome.
In the examination of a great number of samples, a reaction container having multiple V- or U-bottomed wells such as a microplate has been generally used. Also in the examination using such a reaction container, the particle-antibody agglutinate is allowed to precipitate as described above. If the particle-antibody agglutinate is not loosened, then the agglutinate is captured on the bottom face of the reaction container, thereby forming a pattern of the particles spread on the bottom face (positive). On the other hand, if the agglutinate is loosened, then the particle gathers on the lowest region of the bottom face of the reaction container (negative). An intermediate reaction (weakly positive reaction) is detected as an intermediate agglutination pattern. The method enables visual or optical observation of the agglutinate captured on the bottom face of the reaction container microplate from above or below the microplate, and thus it is suitable for the examination of a great number of samples. However, the capturing efficiency of the agglutinate on the microplate bottom face depends significantly on the condition of the bottom face. Although the agglutinate should stably adhere to the bottom face in the examination, a weak agglutinate easily slides from an unprocessed smooth bottom face, indicating a false negative.
As described in Jpn. Pat. Appln. KOKOKU Publication Nos. 61-44268 and 63-60854, studies has been conducted aimed at improving the agglutinate-capturing efficiency by forming unevenness on the bottom face of the reaction container or by roughening the surface of the bottom face. Although there was some improvement in the agglutinate-capturing efficiency by the method, there is a limit on the agglutinate-capturing efficiency only with the bottom face of a well. In addition, blood cells are precipitated spontaneously in the method, and thus the method requires an extended period of time for the examination.
Recently, a container filled with insoluble particles was developed for more efficient capturing of the agglutinate. The passive agglutination method using the container is generally called a microcolumn agglutination method.
As described in Jpn. Pat. Appln. KOKOKU Publication No. 8-7215, Y. Lapierre et al. developed a reaction container, which consists of a card and rod-shaped microreaction tubes formed thereon, wherein the tubes are filled with insoluble particles, such as polymer or glass particles, having a diameter of 10 to 200 μm, and which allows efficient differentiation between agglutinated and unagglutinated red blood cells by centrifugation. A side view of the reaction container is shown in FIG. 12. In addition, EP725276 discloses a similar reaction container filled with glass beads. These devices have a characteristic in that immunologically inactive insoluble particles are used. In both the devices, multiple rod-shaped microreaction tubes filled with insoluble particles are embedded in a plate-shaped plastic plate, and each microreaction tube is used as it is held upright in the vertical direction. During assay, a liquid sample is injected through an inlet on top of the microreaction tube standing in the vertical direction; the container is centrifuged after reaction; and the agglutinate captured by the insoluble particles is detected visually or optically from the side wall. Presence of the agglutinate on the insoluble particles indicates positive reaction; presence of the agglutinate in the intermediate layer of the insoluble particles indicates weak positive reaction; and precipitation of the agglutinate to below the insoluble particles indicates negative reaction.
The rod-shaped microreaction tube described above has advantages that it is possible to complete the reaction in a shorter period of time by using centrifugation, and the evaluation is also easy, because the agglutination pattern differs significantly between the positive and negative reactions. However, because the agglutination pattern develops in the vertical direction and the insoluble particle layer is opaque, it is not possible to examine the reaction in the direction from above or below and thus, the observation is only made through the side wall. Therefore, in observation of the agglutinates on multiple cards, it was necessary to hold up and evaluate each card separately by visually viewing it horizontally, causing a significant obstacle to high-throughput analysis in an automatic analyzer.
To solve the problem above and examine a great number of samples by the microcolumn agglutination method, German Patent No. 10061515 discloses a microplate-shaped container having multiple reaction chambers, of which at least part of the bottom face is narrowed and which is filled with insoluble particles. The container enables acquisition of a flat-spread pattern of the agglutinate when the reaction is positive, and a pattern in which the non-agglutinate gathers in the narrowed region when it is negative, thus allowing observation both from above and below. However, the method still had a problem that it was not possible to detect the pattern accurately in the direction from above or below, especially when the reaction is weakly positive.
Further, Jpn. Pat. Appln. KOKOAI Publication No. 2004-20535 discloses a container allowing detection of the agglutination pattern from the bottom. The cross section of the container is shown in FIG. 13. FIG. 13 shows that, in a transparent container main body 1 having a V-bottomed well 2, a spacer 4 having a lower face facing the bottom face of the well in parallel is placed so that there is a gap layer on a bottom face 3 of the well. Insoluble particles are filled in the gap layer formed between the spacer and the bottom face of the well, forming a fluidal separation layer 5. Presence of the agglutinate on the fluidal separation layer indicates positive reaction; presence of the agglutinate in the intermediate layer of the fluidal separation layer indicates weak positive reaction; and presence of the agglutinate at the bottom of the well indicates negative reaction. It is possible to observe and identify each agglutination pattern in the direction from below the well bottom face 3.
However, it is not easy to form such a V- or U-shaped space while placing a spacer as it floats inside the reaction container, because it requires an additional support material for supporting the spacer. It is also difficult to introduce a sample in the V- or U-shaped space of the reaction container evenly, because the sample inlet is circular in shape. If the sample is introduced therein unevenly, it has an adverse effect on the agglutination pattern. In addition, it is difficult to observe the positive-reaction agglutinate in the container shown in FIG. 13, because the bottom of the transparent container main body 1 has a certain thickness.